Polyvalent immunogen

ABSTRACT

The present invention relates, generally, to a polyvalent immunogen and, more particularly, to a method of inducing neutralizing antibodies against HIV and to a polyvalent immunogen suitable for use in such a method.

This is a continuation-in-part of application Ser. No. 10/289,228, filedNov. 7, 2002, which claims priority from Provisional Application No.60/331,036, filed Nov. 7, 2001, the contents of which are incorporatedherein by reference.

This invention was made with government support under Contract No.W-7405-ENG-36 awarded by the U.S. Department of Energy. The Governmenthas certain rights in the invention.

TECHNICAL FIELD

The present invention relates, generally, to a polyvalent immunogen and,more particularly, to a method of inducing neutralizing antibodiesagainst HIV and to a polyvalent immunogen suitable for use in such amethod.

BACKGROUND

Immunogenic peptides have been developed that elicit B and T cellresponses to various strains of human immunodeficiency virus (HIV)(Palker et al, J. Immunol. 142:3612–3619 (1989), Haynes et al, Trans.Am. Assoc. Physician 106:31–41 (1993), Haynes et al, J. Immunol.151:1646–1653 (1993), Haynes et al, AID Res. Human Retroviruses11:211–221 (1995)) (see also WO 97/14436). These peptides consist of twocomponents, each derived from noncontiguous regions of the HIV gp120envelope protein. One envelope component consists of 16 amino acidresidues from the fourth constant (C4) domain of HIV gp120, and includesa T-helper epitope (Cease et al, Proc. Natl. Acad. Sci. USA 84:4249–4253(1987)). Linked to the carboxyl terminus of this gp120 C4 region peptideis a 23 amino acid segment from the third variable (V3) domain of gp120,that includes a B cell neutralizing antibody epitope for cellline-adapted HIV strains (Palker et al, J. Immunol. 142:3612–3619(1989), (Palker et al, Proc. Natl. Acad. Sci. USA 85:1932–1936 (1988),Rusche et al, Proc. Natl. Acad. Sci. USA 85:3198–3202)), a T-helperepitope (Palker et al, J. Immunol. 142:3612–3619 (1989)), and twocytotoxic T lymphopoietic (CTL) epitopes (Clerici et al, J. Immunol.146:2214–2219 (1991), Safrit et al, 6^(th) NCVDG Meeting, Oct. 30 toNov. 4, 1993)). In mice and rhesus monkeys, these C4-V3 hybrid peptideshave induced antibodies that bind to native gp120 and neutralize theparticular cell line-adapted strain of HIV from which the V3 segment wasderived, as well as induce T helper cell proliferative responses and MHCClass I-restricted CTL responses that kill HIV or HIV protein expressingtarget cells (Palker et al, J. Immunol. 142:3612–3619 (1989), Haynes etal, AID Res. Human Retroviruses 11:211–221 (1995)). Recently, it wasshown that this gp120 peptide design can induce antibodies thatneutralize primary HIV isolates and simian-human immunodeficiencyviruses (SHIV) expressing primary HIV isolate envelopes (Liao et al, J.Virol. 74:254–263 (2000)). Moreover, in a challenge trial of thisimmunogen in rhesus monkeys, it was shown that C4-V3 peptides from thegp120 of the pathogenic SHIV 89.6P, induced neutralizing antibodies thatprevented the fall in CD4 counts after challenge with SHIV 89.6P (Letvinet al, J. Virol. 75:4165–4175 (2001)). Therefore, anti-V3 antibodies canprotect primates against primary isolate SHIV-induced disease.

A prototype polyvalent HIV experimental immunogen comprised of theconserved C4 region of gp120 and the V3 regions of HIV isolates MN,CANO(A), EV91 and RF has been constructed and has been found to behighly immunogenic in human clinical trials (Bartlett et al, AIDS12:1291–1300 (1998), Graham et al, Abstract, AIDS Vaccine (2001)). Thus,understanding secondary and higher order structures of the components ofthis polyvalent immunogen, as well as defining strategies to optimizegp120 immunogen antigenicity, is important to HIV vaccine designefforts. In addition, recent data suggest that the HIV V3 region may beinvolved in regulating gp120 interactions with HIV co-receptors, CXCchemokine receptor 4 (CXCR4) and chemokine receptor type 5 (CCR5)(Berger, AIDS Suppl. A:53–56 (1997)).

In previous studies, nuclear magnetic resonance (NMR) has been used tocharacterize conformations of the multivalent immunogen C4-V3 peptidesin solution (de Lorimier et al, Biochemistry 33:2055–2062 (1994), Vu etal, Biochemistry 35:5158–5165 (1996), Vu et al, J. Virol. 73:746–750(1999)). It as been found that the V3 segments of each of the four C4-V3peptides displayed evidence of preferred solution conformations, withsome features shared, and other features differing among the fourpeptides. The C4 segment, which is of identical sequence in all thepeptides, showed in each case a tendency to adopt nascent helicalconformations (de Lorimier et al, Biochemistry 33:2055–2062 (1994), Vuet al, Biochemistry 35:5158–5165 (1996), Vu et al, J. Virol. 73:746–750(1999)).

The C4 sequence as a peptide does not elicit antibodies that bind nativegp120 (Palker et al, J. Immunol. 142:3612–3619 (1989), Haynes et al, J.Immunol. 151:1646–1653 (1993), Ho et al, J. Virol. 61:2024–2028 (1987),Robey et al, J. Biol. Chem. 270:23918–23921 (1995)). This led to thespeculation that the nascent helical conformations exhibited by the C4segment might reflect a conformation not native to HIV gp120. Amino-acidsequence homology between the gp120 C4 region and a human IgA CH1 domainhas been noted (Maddon et al, Cell 47:333–348 (1986)). By comparison tothe structure of mouse IgA (Segal et al, Proc. Natl. Acad. Sci. USA71:4298–4302 (1974)), the C4-homologous region of IgA has a β strandsecondary structure (de Lorimier et al, Biochemistry 33:2055–2062(1994)). Therefore, while the C4 gp120 peptide in solution adoptsnascent helical conformations, the native structure of this gp120 C4region may be quite different (ie, in the context of gp 120 have a βstrand secondary structure).

The present invention results, at least in part, from the results of astudy with a three-fold purpose. First, C4-V3HIVRF peptides with aminoacid substitutions designed to minimize C4 α-helical peptideconformation and promote β strand C4 secondary structures wereconstructed in order to induce anti-native gp120 antibodies with themodified C4 peptide. Second, tests were made to determine if any ofthese mutated C4-V3RF peptides would enhance gp120 V3 region peptideimmunogenicity, and therefore augment anti-HIVRF gp120 V3 loop antibodyresponses. Finally, the solution conformers of each peptide studiedimmunologically were also solved using NMR to correlate peptideconformers with peptide immunogenicity.

SUMMARY OF THE INVENTION

The present invention relates to a method of inducing neutralizingantibodies against HIV and to peptides, and DNA sequences encoding same,that are suitable for use in such a method.

Objects and advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Summary of antibody binding titers to immunizing peptide after 2or 3 boosts of 3 mice in each group with immunizing peptide. There was aslight enhancement of levels of antibody induced by the E9G variantafter 2 but not 3 boosts, while the E9V variant significantly boostedantibody levels compared to the C4-V3RF(A) peptide after 2 and 3 boosts.Antibody to the K12E variant induced by the K12E peptide wassignificantly lower than C4-V3RF(A) induced antibody levels after both 2and 3 boosts.

FIG. 2: NMR spectra of the four C4-V3RF variant peptides (SEQ ID NOS61–64, respectively, in order of appearance).

FIG. 3: C4_(E9V)-V389.6 peptides bound better to human PB lymphocytesand monocytes than did the C4-V3 89.6 peptides. Similar data wereobtained with the C4-V3 89.6P and C4-E9V-89.6P peptides. Sequence of theC4-V389.6 peptide form H1V89.6 isolate was:KQIINMWQEVGKAMYA-TRPNNNTRRRLSIGPGRAFYARR (SEQ ID NO: 1); the sequence ofthe C4_(EgV)-V389.6 peptide was:KQIINMWQVVGKAMYA-TRPNNNTRRRLSIGPGRAFYARR (SEQ ID NO: 2); the sequence ofthe C4-V389.6P peptide was: KQHNMWQEVGKAMYA-TRPNNNTRERLSIGPGRAFYARR (SEQID NO: 3); the sequence of the C4E9V-V389.6P peptide was:KQIINMWQVVGKAMYA-TRPNNNTRERLSIGPGRAFYARR (SEQ ID NO: 4).

FIG. 4: Neutralization of BAL in PBMC.

FIG. 5: Neutralization of HIV primary isolates by sera from guinea pig(GP) 469 immunized with the C4-V3 peptide 62.19. The isolates tested arelisted on the right side. The grey and white areas indicate noneutralization. The red boxes indicate >50% neutralization. The titersare 1:10, 1:30, 1:90 and 1:270 going across in each column.

FIG. 6: C4-V3 sequences tested (SEQ ID NOS 67–96, respectively, in orderof appearance).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a composition comprising a multiplicityof immunogenic hybrid peptides, each comprising two components. Onecomponent includes a T-helper epitope and can comprise residues from theC4 domain of HIV gp120. The second component comprises residues from theV3 domain of gp120 and includes a B cell neutralizing antibody epitope.

Advantageously, the first component comprises about 16 contiguousresidues from the C4 domain (about residues 421 to 436) and the secondcomponent comprises about 23–25 contiguous residues from the V3 domain(about residues 297 to 322). The components can, however, be longer, andcan comprise, for example, the entirety of the cysteine to cysteine V3loop region, or be shorter. Preferably, the V3 component is linked Cterminal to the C4 component peptide. The hybrid peptides can includeadditional sequences (e.g., linkers (e.g., cysteine, serine or lysinelinkers) between the C4 and V3 components). The composition can, forexample, comprise 5 to 10 hybrid peptides, 10 to 15 hybrid peptides or25 to 30 hybrid peptides. The number of hybrid peptides used willdepend, at least in part, on the target population.

Preferred first components comprising residues from the C4 domain areshown in the Tables that follow (see particularly Tables 6 and 7). OtherT helper determinants from HIV or from non-HIV proteins can also beused. For example, a further T helper epitope suitable for use in theinvention is from HIV gag (e.g., residues 262–278). One such sequence,designated GTH1, is YKRWIILGLNKIVRMYS (SEQ ID NO: 5) (from HIV p24 gag).Variants of this sequence can also be used. Alternatively, or inaddition, a carbohydrate such as the outer membrane protein ofpneumococcus, or another carbohydrate or protein with immunogenic, Thelper activity can be used.

The V3 components of the hybrid peptides present in the instantcomposition are selected so as to be representative of higher orderstructural motifs present in a population, which motifs mediate V3functions in the course of envelope mediated HIV interaction with hostcells. The Los Alamos National Laboratories Human Retroviruses and AIDSDatabase (Human Retroviruses and AIDS, 2000, Published by theTheoretical Biology and Biophysics G T-10, Mail Stop K710, LANL, LosAlamos, N.Mex.) presently contains over 14,000 HIV V3 envelopesequences, showing the extraordinary diversity the virus has obtainedsince originating in man in Africa approximately 50 years ago. Forexample, among 432 HIV-1 V3 sequences derived from individuals infectedwith subtype C (designated “Clade C”) in Africa currently available inthe HIV database, 176 distinct variants of a 23 amino acid stretch atthe tip of the V3 loop have been found. Similarly, among 6870 B subtype(designated “Clade B”) V3 sequences from the US, 1514 unique forms havebeen found.

A method has been developed to organize short antigenic domains byprotein similarity scores using maximum-linkage clustering. This methodenables the visualization of the clustering patterns as a dendrogram,and the splitting patterns in the dendrogram can be used to defineclusters of related sequences (Korber et al, J. Virol. 68:6730–6744(1994)). The method allows the use of several different amino acidsimilarity scoring schemes available in the literature, preferred is theamino acid substitution matrix developed by Henikoff and Henikoff (seeAdvances in Protein Chemistry 54:73–97 (2000) and Proteins: Structure,Function and Genetics 17:49–61 (1993)), designed to give substitutionsthat are well tolerated in conserved protein structural elements a highscore, and a low score to those that are not. Typically excluded fromconsideration very rare, highly divergent peptides, and favored arepeptides found in many individuals within the population. In a selectedset of sequences, most of the unique forms are within one or two aminoacids from a least one other of the peptides chosen. This method hasbeen applied to clustering the large number of variants of the antigenictip of the V3 domain within Clade B and Clade C into groups (about 25)that are likely to be cross-reactive within the group. Based on theseclustering patterns, variants (e.g., about 25–30) are selected that arerepresentative or “central” to each group, for testing for antigenicity.The HIV Clade B and Clade C gp120 envelope V3 sequences have beenanalyzed, as described above, for groups of V3 sequences predicted tohave structural similarities. Twenty five Clade C and 30 Clade B groupshave been defined, and chosen out of each group is a common, or the mostcommon, sequence as a representative of that group. The selected V3sequences have been included in a C4-V3 design thereby providing a 25peptide Clade C immunogen, and a 30 peptide Clade B immunogen (seeTables 6 and 7).

TABLE 6 C4-V3 design of Clade C V3 sequences C4-V3-C1KQIINMWQVVGKAMYA-trpnnntrksirigpGqtfyatg (SEQ ID NO: 6) C4-V3-C2KQIINMWQVVGKAMYA-trpnnntrksirigpGqtfyaRg (SEQ ID NO: 7) C4-V3-C3KQIINMWQVVGKAMYA-trpnnntrksirigpGqtfyaAg (SEQ ID NO: 8) C4-V3-C4KQIINMWQVVGKAMYA-IrpnnntrksVrigpGqtfyatg (SEQ ID NO: 9) C4-V3-C5KQIINMWQVVGKAMYA-trpnnntrksirigpGqtfFatg (SEQ ID NO: 10) C4-V3-C6KQIINMWQVVGKAMYA-trpnnntrksirigpGqtfyatN (SEQ ID NO: 11) C4-V3-C7KQIINMWQVVGKAMYA-trpnnntrEsirigpGqtfyatg (SEQ ID NO: 12) C4-V3-C8KQIINMWQVVGKAMYA-trpnnntrRsirigpGqAfyatg (SEQ ID NO: 13) C4-V3-C9KQIINMWQVVGKAMYA-trpnnntrkGirigpGqtfyatg (SEQ ID NO: 14) C4-V3-C10KQIINMWQVVGKAMYA-trpSnntrksirigpGqAfyatg (SEQ ID NO: 15) C4-V3-C11KQIINMWQVVGKAMYA-trpSnntrksirigpGqtfyatN (SEQ ID NO: 16) C4-V3-C12KQIINMWQVVGKAMYA-trpSnntrEsirigpGqtfyatg (SEQ ID NO: 17) C4-V3-C13KQIINMWQVVGKAMYA-trpnnntrksMrigpGqtfyatg (SEQ ID NO: 18) C4-V3-C14KQIINMWQVVGKAMYA-trpGnntrksMrigpGqtfyatg (SEQ ID NO: 19) C4-V3-C15KQIINMWQVVGKAMYA-trpGnntrksirigpGqtLyatg (SEQ ID NO: 20) C4-V3-C16KQIINMWQVVGKAMYA-VrpnnntrksVrigpGqtSyatg (SEQ ID NO: 21) C4-V3-C17KQIINMWQVVGKAMYA-trpGnntrRsirigpGqtfyatg (SEQ ID NO: 22) C4-V3-C18KQIINMWQVVGKAMYA-IrpGnntrksVrigpGqtfyatg (SEQ ID NO: 23) C4-V3-C19KQIINMWQVVGKAMYA-trpnnntrksirigpGqAfyatN (SEQ ID NO: 24) C4-V3-C20KQIINMWQVVGKAMYA-trpnnntrQsirigpGqAfyatK (SEQ ID NO: 25) C4-V3-C21KQIINMWQVVGKAMYA-trpGnntrksirigpGqAfFatg (SEQ ID NO: 26) C4-V3-C22KQIINMWQVVGKAMYA-trpGnntrksVrigpGqAfyatN (SEQ ID NO: 27) C4-V3-C23KQIINMWQVVGKAMYA-trpnnntrkGiHigpGqAfyaAg (SEQ ID NO: 28) C4-V3-C24KWIINMWQVVGKAMYA-trpnnntrkGiGigpGqtfFatE (SEQ ID NO: 29) C4-V3-C25KQIINMWQVVGKAMYA-trpGnntrEsiGigpGqAfyatg (SEQ ID NO: 30)

TABLE 7 C4-V3 peptides Clade B C4-V3-396.2KQIINMWQVVGKAMYA-RPNNNTRRNIHIGLGRRFYAT-* (SEQ ID NO: 31) C4-V3-170.6KQIINMWQVVGKAMYA-RPNNNTRRSVRIGPGGAMFRTG* (SEQ ID NO: 32) C4-V3-82.15KQIINMWQVVGKAMYA-RPNNNTRRSIPIGPGRAFYTTG* (SEQ ID NO: 33) C4-V3-144.8KQIINMWQVVGKAMYA-RPDNNTVRKIPIGPGSSFYTT-* (SEQ ID NO: 34) C4-V3-23.38KQIINMWQVVGKAMYA-RPIKIERKRIPLGLGKAFYTTK* (SEQ ID NO: 35) C4-V3-365.2KQIINMWQVVGKAMYA-RPSNNTRKGIHLGPGRAIYATE* (SEQ ID NO: 36) C4-V3-513.2KQIINMWQVVGKAMYA-RPSNNTRKGIHMGPGKAIYTTD* (SEQ ID NO: 37) C4-V3-1448.1KQIINMWQVVGKAMYA-RPGNTTRRGIPIGPGRAFFTTG* (SEQ ID NO: 38) C4-V3-69.18KQIINMWQVVGKAMYA-RPNNNTRKSIRIGPGRAVYATD* (SEQ ID NO: 39) C4-V3-146.8KQIINMWQVVGKAMYA-RPGNNTRRRISIGPGRAFVATK* (SEQ ID NO: 40) C4-V3-113.1KQIINMWQVVGKAMYA-RPNNNTRRSIHLGMGRALYATG-* (SEQ ID NO: 41) C4-V3-51.23KQIINMWQVVGKAMYA-RPSNNTRRSIHMGLGRAFYTTG-* (SEQ ID NO: 42) C4-V3-72.18KWIINMWQVVGKAMYA-RPNNNTRKGINIGPGRAFYATG-* (SEQ ID NO: 43) C4-V3-36.29KWIINMWQVVGKAMYA-RPNNNTRKGIHIGPGRTFFATG-* (SEQ ID NO: 44) C4-V3-70.18KWIINMWQVVGKAMYA-RPNNNTRKRIRIGHIGPGRAFYATG* (SEQ ID NO: 45) C4-V3-89.14KWIINMWQVVGKAMYA-RPSINKRRHIHIGPGRAFYAT-* (SEQ ID NO: 46) C4-V3-163.7KWIINMWQVVGKAMYA-RLYNYRRKGIHIGPGRAIYATG* (SEQ ID NO: 47) C4-V3-57.20KWIINMWQVVGKAMYA-RPNRHTGKSIRMGLGRAWHTTR* (SEQ ID NO: 48) C4-V3-11.85KWIINMWQVVGKAMYA-RPNNNTRKSINIGPGRAFYTTG---* (SEQ ID NO: 49) C4-V3-34.29KWIINMWQVVGKAMYA-RPNNNTRKSIQIGPGRAFYTTG---* (SEQ ID NO: 50) C4-V3-1.481KWIINMWQVVGKAMYA-RPNNNTRKSIHIGPGRAFYTTG---* (SEQ ID NO: 51) C4-V3-85.15KWIINMWQVVGKAMYA-RPNNNTRKSIHIAPGRAFYTTG---* (SEQ ID NO: 52) C4-V3-62.19KWIINMWQVVGKAMYA-RPNNNTRKSIHIGPGRAFYATE------* (SEQ ID NO: 53)C4-V3-125.9 KWIINMWQVVGKAMYA-RPNNNTRRRISMGPGRVLYTTG* (SEQ ID NO: 54)C4-V3-35.29 KWIINMWQVVGKAMYA-RPNNNTRKRISLGPGRVYYTTG* (SEQ ID NO: 55)C4-V3-74.17 KWIINMWQVVGKAMYA-RPNNNTRKRMTLGPGKVFYTTG* (SEQ ID NO: 56)C4-V3-46.26 KWIINMWQVVGKAMYA-RPDNTIKQRIIHIGPGRPFYTT-* (SEQ ID NO: 57)C4-V3-122.9 KWIINMWQVVGKAMYA-RPNYNETKRIRIHRGYGRSFVTVR* (SEQ ID NO: 58)C4-V3-162.7 KWIINMWQVVGKAMYA-RPGNNTRGSIHLHPGRKFYYSR* (SEQ ID NO: 59)C4-V3-3.323 KWIINMWQVVGKAMYA-RPNNNTRKSINMGPGRAFYTTG (SEQ ID NO: 60)

While the above is offered by way of example, it will be appreciatedthat the same analyses can by performed for HIV Clades A, D, E, F, G, H,M, N, O, etc, to design V3 immunogens that react with HIV primaryisolates from these Clades.

In addition to the sequences described in Tables 6 and 7, a substitutionhas been made in the C4 sequence at position 9 from E to V to enhancethe binding of the C4 region to human immune cell membranes, and toincrease immunogenicity (see Example that follows). Substituting V for Eat position 9 of C4 results in the C4-E9V-V3RF(A) peptide inducing 2–3logs higher anti-gp 120 V3 region antibody levels compared with theoriginal C4-V3RFA(A) peptide. The effect of the E9V substitution is notspecies specific. While not wishing to be bound by theory, the data mayindicate that the ability of the E9V variant peptide to enhance B cellantibody production is not MHC specific but rather it relates in somemanner to non-MHC specific factors, such as the ability of the peptidesto bind to the lipid bilayer of immune cells. The data presented in FIG.3 demonstrate the ability of C4_(E9V)-V389.6 peptides to bind to humanPB lymhocytes and monocytes. The ability of the C4 and C4E9V “T helper”determinants to facilitate immunogenicity of the V3 region may be due tothe ability of helical amphipathic structures to interact with lipidbilayers in a non-MHC related manner and promote peptideinternalization. The invention encompasses the use of C4 sequences inaddition to those described above.

In addition to the composition described above, the inventionencompasses each of the hybrid peptides disclosed as well as each of thecomponents (C4 and V3), alone or in covalent or non-covalent associationwith other sequences, as well as nucleic acid sequences encoding any andall such peptides. The invention provides an HIV immunogen that caninduce broadly reactive neutralizing antibodies against HIV of multiplequasispecies, and across clades. With reference to Example 3, the “dualD” HIV isolate, neutralized by serum from GP 469 immunized with peptide62.19 to a titer of 1:30, is a Clade A/G recombinant HIV isolate. Thisdemonstrates that this peptide (62.19), for example, can induceantibodies against a non-B HIV isolate. The 62.19 and other V3 sequencesin FIG. 6 and Tables 10 and 11 can be expressed either alone or, forexample, as a C4-V3 sequence, as in FIG. 6. It will be appreciated thatthe same analysis described in Example 3 can by performed for any of HIVClades A, D, E, F, G, H, M, N, O, etc, to identify V3 immunogens thatreact with HIV primary isolates from one or more of these Clades.

The peptide immunogens of the invention can be chemically synthesizedand purified using methods which are well known to the ordinarilyskilled artisan. (See, for example, the Example that follows.) Thecomposition can comprise the peptides linked end to end or can comprisea mixture of individual peptides. The peptide immunogens can also besynthesized by well-known recombinant DNA techniques. Recombinantsynthesis may be preferred when the peptides are covalently linked.Nucleic acids encoding the peptides of the invention can be used ascomponents of, for example, a DNA vaccine wherein the peptide encodingsequence(s) is/are administered as naked DNA or, for example, a minigeneencoding the peptides can be present in a viral vector. The encodingsequence(s) can be present, for example, in a replicating ornon-replicating adenoviral vector, an adeno-associated virus vector, anattenuated mycobacterium tuberculosis vector, a Bacillus Calmette Guerin(BCG) vector, a vaccinia or Modified Vaccinia Ankara (MVA) vector,another pox virus vector, recombinant polio and other enteric virusvector, Salmonella species bacterial vector, Shigella species bacterialvector, Venezuelean Equine Encephalitis Virus (VEE) vector, a SemlikiForest Virus vector, or a Tobacco Mosaic Virus vector. The encodingsequence(s), can also be expressed as a DNA plasmid with, for example,an active promoter such as a CMV promoter. Other live vectors can alsobe used to express the sequences of the invention. Expression of theimmunogenic peptides of the invention can be induced in a patient's owncells, by introduction into those cells of nucleic acids that encode thepeptides, preferably using codons and promoters that optimize expressionin human cells. Examples of methods of making and using DNA vaccines aredisclosed in U.S. Pat. Nos. 5,580,859, 5,589,466, and 5,703,055.

The composition of the invention comprises an immunologically effectiveamount of the peptide immunogens of this invention, or nucleic acidsequence(s) encoding same, in a pharmaceutically acceptable deliverysystem. The compositions can be used for prevention and/or treatment ofimmunodeficiency virus infection. The compositions of the invention canbe formulated using adjuvants, emulsifiers, pharmaceutically-acceptablecarriers or other ingredients routinely provided in vaccinecompositions. Optimum formulations can be readily designed by one ofordinary skill in the art and can include formulations for immediaterelease and/or for sustained release, and for induction of systemicimmunity and/or induction of localized mucosal immunity (e.g, theformulation can be designed for intranasal administration). The presentcompositions can be administered by any convenient route includingsubcutaneous, intranasal, oral, intramuscular, or other parenteral orenteral route. The immunogens can be administered as a single dose ormultiple doses. Optimum immunization schedules can be readily determinedby the ordinarily skilled artisan and can vary with the patient, thecomposition and the effect sought. By way of example, it is noted thatapproximately 50 μg–100 μg of each hybrid peptide can be administered,for example, intramuscularly (e.g. 3×).

The invention contemplates the direct use of both the peptides of theinvention and/or nucleic acids encoding same and/or the peptidesexpressed as minigenes in the vectors indicated above. For example, aminigene encoding the peptides can be used as a prime and/or boost.Importantly, it has been recently shown that recombinant gp120 is notefficacious as a vaccine for HIV in phase III trials (Elias, P., DurhamMorning Herald, Feb. 25, 2003; VaxGen News Conference, Feb. 24, 2003).Thus, it would be advantageous to express, for example, the 62.19 V3loop and/or other V3 loops in Table 11 in the context of gp120 moleculesor gp160 or gp140 molecules, either as expressed soluble recombinantproteins, or expressed in the context of one of the vectors describedabove. This strategy takes advantage of the ability to express native V3conformations within a whole gp120 or gp140 or gp160 HIV envelopeprotein.

One of the preferred gp120, gp140 or gp160 envelopes that, for example,62.19 V3 loops can be expressed with is that of consensus or ancestralHIV envelope artificial sequences (Gaaschen et al, Science 296:2354–2360(2002)). Although artificial and computer designed, one such sequence(the consensus of consensus envelope) gp120 (con 6) has been shown tobind soluble CD4 and anti-gp120 mabs A32, 1b12, 2G12. After binding mabA32 or soluble CD4, the con 6 gp120 binds the CCR5 binding site mab176—indicating a “native” gp120 conformation.

Thus, the entire V3 loops from the Los Alamos Database from thesequences of one or more of the peptides in Table 11 can be expressed inthe consensus (con 6) or other consensus or ancestral gp120, gp140, orgp160 envelope protein, or expressed in a native gp120, gp140, or gp160,such as HIV BAL or HIV JRFL, and used as an immunogen as a recombinantenvelope protein, or used as an immunogen expressed in one of thevectors above.

The V3 peptides or recombinant proteins can be used as primes or boostswith the V3 peptides or recombinant gp120s, gp140s or gp160s expressedin the above vectors used as primes or boosts.

A preferred immunogen is the consensus 6 gp120 expressing thefull-length 62.19 V3 loop, expressed as a DNA plasmid as a primaryimmunization, followed by adenovirus expressing the Con 6 envelopeexpressing the 62.19 V3 sequence from the Los Alamos Database as abooster immunization.

Certain aspects of the invention can be described in greater detail inthe non-limiting Example that follows.

EXAMPLE 1

Experimental Details

Peptide Design, Synthesis and Purification.

Peptides were designed, as shown in Table 1. It was hypothesized thatalteration of the C4 sequence to reduce its helical conformationaltendency in peptides might cause enrichment of solution conformersresembling a β strand conformation. This in turn might cause C4 to beimmunogenic for antibodies recognizing the native conformation of the C4(part of the CD4 binding site) region of gp120. The present workdescribes tests of this hypothesis in chimeric peptide C4-V3 RF, whichhas a V3 segment from gp120 of HIV strain RF, and three sequencevariants wherein single amino-acid replacements have been introduced atposition 9 in the C4 segment, Glu (E) to Gly (G), Glu (E) to Val (V),and at position 12, Lys (K) to Glu (E) (Table 1). These replacementswere made in part to disrupt possible stabilization of helicalconformations due to side-chain (i, i+3) charge interaction between E9and K12 (Scholtz et al, Biochemistry 32:9668–9676 (1993)). In addition,the substitution in C4_(E9G)-V3RF(A) was expected to disfavor helixformation by introducing greater main-chain flexibility (Chakrabartty etal, Adv. Protein Chem. 46:141–176 (1995)). Furthermore the substitutionin C4_(E9G)-V3RF(A) introduced two adjacent valine residues which hasbeen hypothesized to favor extended conformations. Thus, the parentpeptide, C4-V3RF(A) (Haynes et al, AID Res. Human Retroviruses11:211–221 (1995)) contained 16 N-terminal residues from the C4 domainof gp120_(IIIB) and 23 C-terminal residues from the V3 domain of gp120of HIVRF.

TABLE 1 Peptides Used in This Study Sequence Peptide      C4       V31             16 17                   39 C4- KQIINMWQEVGKAMYATRPNNNTRKSITKGPGRVIYATG V3RF(A) (SEQ ID NO: 61) C4_(E9G)-KQIINMWQGVGKAMYA TRPNNNTRKSITKGPGRVIYATG V3RF(A) (SEQ ID NO: 62)C4_(E9V)- KQIINMWQVVGKAMYA TRPNNNTRKSITKGPGRVIYATG V3RF(A) (SEQ ID NO:63) C4_(K12E)- KQIIINMWQEVGEAMYA TRPNNNTRKSITKGPGRVIYATG V3RF(A) (SEQ IDNO: 64) All sequences from Los Alamos National Laboratory AIDS SequenceDatabase.

Peptides were synthesized by fluorenylmethoxycarbonyl chemistry on anABI 43 1A peptide synthesizer (Applied Biosystems, Inc., Foster City,Calif.), then purified by reverse-phase high performance liquidchromatography. The purity and identity of the product were confirmed bydetermining molecular mass by electrospray mass spectrometry.

Immunization Methods.

Mice were immunized with 50 μg of the indicated peptide in incompleteFreund's adjuvant (1SA51, Seppic Inc., Paris France) at weeks 0, 3, and7 and bled at weeks 2, (bleed 1 after boost 1), week 5 (bleed 2 afterboost 2) and week 8 (bleed 3 after boost 3). Immune responses were seenafter bleed 2 in most animals and data are reported from bleeds 2 and 3.

Guinea pigs were immunized intranasally with 200 μg of C4-V3 peptide insaline with 1 μg of cholera toxin as adjuvant as described. Guinea pigswere immunized on day 0, day 14 and day 21 and serum samples before and1 week following each immunization obtained by cardiac puncture.

ELISA Assay.

Anti-HIV env peptide ELISA assays were performed as previously described(Haynes et al, J. Immunol. 151:1646–1653 (1993), Haynes et al, AID Res.Human Retroviruses 11:211–221 (1995)).

Splenocyte Proliferation Assay.

Mouse splenocyte proliferation assay using ³H-thymidine incorporationwas performed as previously described (Haynes et al, AID Res. HumanRetroviruses 11:211–221 (1995)).

Neutralizing Antibody Assays.

Assays for ability of anti-HIV antisera to neutralize HIV were performedas described (Palker et al, J. Immunol. 142:3612–3619 (1989), Haynes etal, Trans. Am. Assoc. Physician 106:31–41 (1993), Haynes et al, J.Immunol. 151:1646–1653 (1993), Haynes et al, AID Res. Human Retroviruses11:211–221 (1995)).

NMR Spectroscopy.

Peptides were dissolved to 4 mM in a solution of 90% ¹H₂O, 10% ²H₂O, 20mM NaCl, 5 mM KH₂PO₄, 1 mM sodium azide, 0.5 mM sodium3-(trimethylsilyl) propionate, at a pH of 4.2. The methyl resonance ofthe latter component served as a chemical shift reference.

Spectra of samples prepared in this way were acquired with a VarianUnity 500 MHz spectrometer at a temperature of 278 K. The lock signalwas from deuterium in the sample. The following two-dimensional spectrawere obtained: (a) double-quantum-filtered correlation spectroscopy(DQF-COSY) (Piantini et al, J. Am. Chem. Soc. 104:6800–6801 (1982),Rance et al, Biochem. Biopjys. Res. Commun. 117:479–485 (1983)); (b)total correlation spectroscopy (TOCSY) (Bax et al, J. Magn. Reson.65:355–360 (1985), Levitt et al, J. Magn. Reson. 47:328–330 (1982)) witha mixing time of 150 ins; and (c) nuclear Overhauser exchangespectroscopy (NOESY) (Jeener et al, J. Phys. Chem. 71:4546–4553 (1979))with a mixing time of 300 ins. Water resonance was suppressed byselective saturation during the relaxation delay, and, for NOESY, duringthe mixing period. The spectral width was 6700 Hz, with the indirectlyacquired dimension collected as 750 (COSY), 512 (TOCSY), or 350 (NOESY)complex increments; and the directly acquired dimension containing 1024complex points. Data were processed with FELIX 2.3 software (Biosym, SanDiego, Calif.). Directly acquired free-induction decays were correctedfor base-line offset. Decays in both dimensions were multiplied by asinebell-squared function (phase shifted by 75°) and zero-filled to 2048points before Fourier-transformation.

Peptide Membrane Binding Assay.

Peptides at 100 ng/ml were incubated with 106 peripheral bloodmononuclear cells for 1 hour at 4° C., washed ×3 with phosphate bufferedsaline PHz 7.0, contained 0.1% sodium azide, then incubated guinea piganti-HIV 89.6 V3 antisera (×1 hr) (Liao et al, J. Virol. 74:254–263(2000)), wash as above and then incubated with FITC-conjugated goatanti-guinea pig IgG. After a final wash as alone, the cells wereanalyzed for the relative amount of peptide bound to either PBlymphocytes or PB monocytes as reflected in the mean fluorescent channel(MFC) of reactivity of the anti-HIV 89.6 V3 antisera.

Results

Anti-gp120 V3 Antibody Responses Following Immunization of Mice WithC4-V3RF, C4_(E9V)-V3RF(A), C⁴ _(E9G)-V3RF(A) and C4_(K12E)-V3RF(A)Peptides.

First, the ability of C4-V3HIVRF variants to modulate the immunogenicityof the peptide with regard to antibodies to the V3 portion of the C4-V3immunogen were assayed. The results (FIG. 1, Table 2) show differencesamong the four peptides in their ability to induce anti-HIVRF V3antibody responses. Sera from C4_(E9V)-V3RF(A)-immunized mice had a loghigher anti-V3 antibody titer than either mice immunized with the nativeC4-V3RF(A) peptide or the C4E9V-V3RF(A) peptide variant. After oneimmunization, no anti-V3RF antibody response was seen in mice immunizedwith either C4-V3RF(A), C4_(E9G)-V3RF(A), or C4_(K12E)-V3RF(A) peptides.However, after only one immunization with 50 μg of the C4_(E9V)-V3peptide, the geometric mean titer to V3RF(A) peptide was 1:5012 (n=3mice), with titers of 1:3200, 1:3200 and 1:12,800 in each of the threemice tested, respectively. Thus, the E9V C4-V3RF(A) variant induced ahigher titer and earlier anti-gp 120 V3 antibody responses than theother C4-V3RF(A) peptides tested. After 2 boosts,C4_(E9V)-V3RF(A)-immunized mice had 2 logs higher anti-V3 antibodyresponses than did C4-V3RF(A) immunized mice (FIG. 1, Table 2).

TABLE 2 Comparison of the Ability of C4-V3 Peptides To Induce HIV gp120Anti-C4 and Anti-V3 Antibodies in Balb/c Mice Number of Peptide on Platein ELISA For Anti-Peptide Antibody Assay Peptide Animals C4 ImmunogenGeometric Mean Titer V3RF(A) C4-V3RF(A) C4E9G-V3RF(A) C4E9V-V3RF(A)C412EV3RF(A) C4-V3RF(A) 6 2 1,584 2,239 1,195 1,584 1,412C4_(E9G)-V3RF(A) 6 2 6,310 7,079 5,623 3,162 3,548 C4_(E9V)-V3RF(A) 5 14151,356 131,825 87,096 87,096 114,815 C4_(K12E)-V3RF(A) 6 1 8 8 1 3 3Data represent the reciprocal of endpoint dilutions at which the E/C was3.0 in anti-peptide ELISA after two immunizations.

The C4_(K12E)-V3RF(A) peptide variant induced anti-V3 antibody responses3 logs lower than the C4-V3RF(A) peptide after 2 immunizations (FIG. 1,Table 2). Thus, single amino-acid replacements in the C4 T helper regionhad extraordinary effects on immunogenicity of the HIVRF gp120 V3domain.

Comparison of the Ability of C4-V3RF(A) Peptides to Induce Anti-HIVgp120Peptide 3H-Thymidine Incorporation in Splenocytes from Naive andPeptide-Immunized Mice.

Next, C4-V3 peptides were tested for their ability to stimulateproliferation of splenocytes from peptide-immunized mice. Balb/c micewere sacrificed after the third peptide immunization and theirsplenocytes assayed for the ability to proliferate to PHA and to eachpeptide type (Table 3). It was found that C4-V3RF(A), C4_(E9V)-V3RF(A),and C4_(K12E)-V3RF(A) peptides all induced in vitro proliferativeresponses to the immunizing peptides, whereas the C4_(E9G)-V3RF(A)variant peptide did not induce proliferative responses in E9G-primedmice significantly over responses of naive mice (Table 3). Regarding theability of the E9V peptide variant to induce earlier and greater anti-V3antibody responses compared to the other peptides tested, theC4_(E9V)-V3RF(A) peptide-primed splenocytes for proliferation to theimmunizing peptide only minimally better than did each of the otherthree peptides (Table 3). Thus, altered induction of T helper cellproliferative responses did not explain the differences in peptideimmunogenicity.

TABLE 3 Comparison of the Ability of C4-V3 Peptides To Induce Anti-HIVgp120 Peptide ³H-Thymidine Incorporation in Splenocytes from Naïve andImmunized Mice Peptide Used As Stimulator in 3H-Thymidine IncorporationAssay Peptide Mean ± SEM CPM per 10⁶ Splenocytes in Culture Immunogen NC4 V3RF(A) C4-V3RF(A) C4_(E9G)-V3RF(A) C4_(E9V)-V3RF(A)C4_(K12E)-V3RF(A) None (Naïve 6 613 ± 322 408 ± 140 149 ± 84  114 ± 85 74 ± 47 187 ± 165 Balb/c) C4-V3RF(A) 6 2,289 ± 955 ± 353 8,390 ± 8,067 ±1,728 6,242 ± 1,787 6,198 ± 1,343 1,332 1,424^(a) C4_(E9G)-V3RF(A) 6 408± 95  708 ± 325 2,103 ± 1,170   3,559 ± 2,310^(b) 988 ± 340 1,101 ±399   C4_(E9V)-V3RF(A) 5 84 ± 52 1,463 ±   933 ± 4,528 11,743 ± 3,830  24,824 ± 5,581^(c) 10,269 ± 3,592    473 C4_(K12E)-V3RF(A) 6 3,430 ±4,417 ± 8,670 ± 3,865 13,237 ± 7,513 ± 2,951 12,644 ± 2,796 2,217 8,5634,138^(d) Data represent peak 3H-thymidine responses at 7 days. CPM =CPM experimental − experimental − experimental control. ^(a)p < .001 vsnaïve mice; p = NS vs C4-V3RF(A) or C4K12E-V3RF(A) stimulatedC4K12E-V3RF(A) immunized splenocytes. ^(b)p = NS vs naïve mice. ^(c)p <.001 vs naïve mice. ^(d)p < .02 vs naïve mice.

The lower antibody titer induced by the C4_(K12E)-V3 peptide againstV3RF(A) was not an artifact attributable to lack of ability of the V3peptide not binding to the ELISA plate, as sera fromC4_(E9V)-V3RF(A)-induced antisera had high reactivity to the V3RF(A)peptide on the ELISA plate. Similarly, the C4_(K12E)-V3RF(A) peptidecould bind anti-V3RF antibody, as multiple antisera raised against C4-V3peptides bound the C4_(K12E)-V3 variant (Table 2).

Antibody levels to the C4 region were also tested. The C4 region inducedonly a minimal antibody response compared to the V3 region, with all theC4-V3 peptides tested (Table 2).

Anti-gp 120 V3 Antibody Responses Following Immunization of Guinea Pigs.

Next, 2 guinea pigs were immunized each with 200 μg of C4-V3RF(A),C4_(E9G)-V3 RF(A), C4_(E9V)-V3 RF(A) or C4_(K12E)-V3 RF(A) peptideintranasally with 1 μg cholera toxin adjuvant in saline. Intranasalimmunization of peptides with cholera toxin has been previously shown toresult in CTL and titers of anti-peptide antibody similar in levels totiters induced by initial antigens administered subcutaneously orintramuscularly in oil in water adjuvants such as complete andincomplete Freund's adjuvant. In addition, it was desirable to determinethe ability of C4-V3 peptides in an aqueous solution (such as in salinefor intranasal immunization) to induce anti-HIV antibody responses inorder to correlate reactivity of antibodies generated against peptide inan aqueous adjuvant with peptide conformers solved in an aqueoussolution. Finally, there was interest in determining if the amino acidsubstitutions in the C4 region conferred on the C4-V3 peptides the samepattern of immunogenicity as seen in oil in water adjuvant in mice.

It was found that after 2 immunizations the C4-V3 RF(A) peptide induceda mean anti-HIV peptide antibody titer of 3981, peptide induced titersof 1 log (GMT=31,623) higher. As in mice, substituting the Glu (E) forLys (K) at position 12 in the C4 peptide abrogated peptideimmunogenicity in guinea pigs (GMT=16) (Table 4).

TABLE 4 Titers of C4-V3 HIV Envelope Antibodies Induced by C4-V3RF(A)Peptides in Guinea Pigs Immunizing Peptide Titer Against ImmunizingPeptide* C4-V3RF(A) 3,981 C4-_(E9G)-V3RF(A) 2,818 C4-_(E9V)-V3RF(A)31,623 C4-_(K12E)V3RF(A) 16 *Data represent the mean titers from 2animals after 2–3 immunizations intranasally with 400 ug of theindicated peptide formulated in saline with cholera toxin as anadjuvant.Ability of Antibodies Against C4-V3 Peptides to Induce NeutralizingAntibodies.

In order to induce high levels of neutralizing antibodies with C4-V3peptides, usually 5 immunizations are given (Palker et al, J. Immunol.142:3612–3619 (1989), Haynes et al, J. Immunol. 151:1646–1653 (1993),Palker et al, Proc. Natl. Acad. Sci. USA 85:1932–1936 (1988), Liao etal, J. Virol. 74:254–263 (2000)). The guinea pig sera from theexperiment presented in Table 4 were tested for ability to neutralizeHIVRF. It was found that one sera from the C4-V3RF(A)-immunized animals(after 3 injections) had a neutralizing antibody titer of 1:40 againstHIVRF, while one animal of the C4_(E9V)-V3RF(A)-injected animals had aneutralizing titer of 1:340 after only 2 injections. Thus, antibodiesinduced by the C4_(E9V)-V³RF(A) peptide can bind to native gp120 andneutralize HIVRF.

Inability of the C4-E9V-RF(A) Sera to Bind to gp120 from HIV_(IIIB).

The V3 loop sequence of HIV_(IIIB) is different from that of HIVRF, andthus HIVRF anti-V3 neutralizing antibodies do not neutralize HIV_(IIIB).To determine if any antibodies were generated by any of the C4-V3RF(A)variant peptides, all the mouse sera in Table 2 were tested, as were theguinea pig sera in Table 4, for the ability to bind to nativerecombinant HIV_(IIIB) gp120 in ELISA. Since anti-HIVRF V3 antibodies donot bind to the HIV_(IIIB) V3 loop, any binding activity of theseanti-C4-V3 sera would be to the C4 region of HIV_(IIIB), which isconserved between HIV_(IIIB) and HIVRF. No binding of any mouse orguinea pig anti-C4-V3 sera to HIV_(IIIB) gp120 was seen, indicating theinability of these peptides to induce antibodies against the nativegp120 C4 region.

Conformational Propensities of C4-V3 RF Sequence Variants in AqueousSolution.

Next, the peptides were examined by NMR to determine whetherconformational changes had been induced by amino-acid sequencealteration.

It was hypothesized that specific amino-acid substitutions in the C4segment would lead to a decrease in the tendency of this region to adopttransient helical conformations. To test this hypothesis, each of thefour peptides, C4-V3RF and variants E9G, E9V and K12E, was subjected to¹H NMR spectroscopy to assign resonances and to analyze nuclearOverhauser effects between hydrogen nuclei on separate residues.

Resonance assignments for nearly all ¹H were determined from TOCSY,DQF-COSY, and NOESY spectra by standard methods (Wuthrich, NMR ofProteins and Nucleic Acids, John Wiley and Sons, New York (1986)), andare shown in FIG. 2. The value of the chemical shift for a main-chain¹H, for example, the a carbon C^(a)H, is correlated with secondarystructure in the case of proteins or well structured peptides (Wishartet al, J. Mol. Biol. 222:311–333 (1991)). Hence, strong tendencies amongC4-V3RF peptides to adopt secondary structure in solution may bemanifested in chemical shift values. This was examined by calculatingfor each peptide the difference in chemical shift between the C—H ofeach residue and a shift value representing the average for allsecondary structures in proteins (Wishart et al, J. Mol. Biol.222:311–333 (1991)). In no peptide were there stretches of sequence withhigh or low values of the chemical shift difference that would beevidence of stable secondary structure, for example helix or β strand.

NMR parameters such as chemical shift and coupling constants are ofteninsensitive indicators of weak preferences for particular conformationssince their values are the average of the entire population, thusobscuring the contribution of a slight bias for populating certainconformations. The nuclear Overhauser effect (NOE) is often moresensitive at revealing conformational propensities because it may giverise to a unique signal, although weak, on a background consisting onlyof random noise. Hence, NOESY spectra of C4-V3RF and its variants werecharacterized to identify each signal and evaluate its relativeintensity. Sequential and medium range NOEs involving main-chain NH orCaH are listed in FIG. 2. These NOEs and the possible conformationalpropensities they represent are discussed as follows forC4_(E9G)-V3RF(A) and C4_(E9V)-V3RF(A). Variant C4_(K12E)-V3RF(A)K12E isdiscussed separately below because it was studied under differentconditions.

In terms of overall conformation, all four peptides showed NOE patternssuggesting no tendency to adopt stable structure. For example,sequential daN(i, i+1) and dNN(i, i+1) NOEs were usually both presentfor each sequential pair of residues, with the former typically moreintense, indicating that f and j main-chain dihedral bond angles variedand maintained on average an extended conformation (Dyson et al, Ann.Rev. Biophys. Chem. 20:519–538 (1991)). Also the absence of long rangeNOEs [(i, i+5) or greater] and the few and generally weak medium-rangeNOEs suggested no significant population of higher order structure.

However, the fact that some medium range NOEs were detected is evidenceof propensity to adopt non-random conformnations in certain regions(Dyson et al, Ann. Rev. Biophys. Chem. 20:519–538 (1991)). Although onlyone mixing time was used for NOESY spectra (300 ins), previous studiesof a related C4-V3 RF peptide (de Lorimier et al, Biochemistry33:2055–2062 (1994)) showed that medium range NOEs were still observableat shorter (75 and 150 ins) mixing times. Hence, the NOEs indicatingmedium range interactions are not likely due to spin-diffusion.

Within the C4 segment C4-V3RF and C4_(E9V)-V3RF(A) showed numerousmedium range NOEs which are consistent with a tendency of this region topopulate nascent helical conformations. The presence of contiguous oroverlapping daN(i,i+2) NOEs from Trp⁷to Tyr¹⁵ (C4-V3RF) and from Ile⁴ toLys12 (E9V) indicates a propensity for nascent helical turns in theseregions (Dyson et al, Ann. Rev. Biophys. Chem. 20:519–538 (1991), Dysonet al, J. Mol. Biol. 201:201–217 (1988)). A dNN(i,i+2) NOE in thisregion in C4-V3 RF (between Lys12 and Met 4) is also consistent withmain-chain f and j dihedral angles representative of helical turns(Dyson et al, Ann. Rev. Biophys. Chem. 20:519–538 (1991)). C4-V3 RFshows three consecutive daN(i,i+3) NOEs from residues Val¹⁰ to Tyr¹⁵,which is highly indicative of full helical turns. The presence ofequivalent NOEs in E9V could not be ascertained due to overlap withother NOEs. However both C4-V3RF and E9V show two dab(i,i+3) NOEs,between Val¹⁰ and Ala¹³ and between Ala¹³ and Met¹⁴. This type of NOE isalso highly suggestive of full helical turns in these regions of C4.

Variant C4_(E9G)-V3RF(A)on the other hand showed no evidence, in termsof medium range NOEs, for preferential population of certainconformations in C4. This absence of medium range NOEs was not duemerely to ambiguities caused by signal overlap, because there were atleast five positions where an NOE was unambiguously absent inC4_(E9G)-V3RF(A), but present in the parent peptide C4-V3 RF. Thus, theE to G substitution in the C4 peptide appeared to prevent helicalconformer formation in the peptide.

In the V3 segment of the three peptides, C4-V3 RF, C4_(E9G)-V3RF(A) andC4_(E9V)-V3RF(A), were medium range NOEs suggesting preferred solutionconformations in certain RE regions. All three peptides showed evidenceof a reverse turn in the sequence Arg¹⁸-Pro¹⁹-Asn²⁰-Asn²¹ (SEQ ID NO:65), where these residues comprised positions 1 to 4, respectively, ofthe turn. The NOE pattern consistent with a reverse turn included a weakdNd(i,i+1) between Arg¹⁸ and Pro¹⁹, undetectable ddN(i,i+1) betweenPro¹⁹ and Asn²¹, weak dad(i,i+1) between Arg¹⁸ and Pro¹⁹strongdaN(i,I+1) between Pro¹⁹ and Asn²⁰, and detectable daN(i,i+2) betweenPro¹⁹ and Asn²⁰ (Dyson et al, J. Mol. Biol. 201:161–200 (1988)). Thedetection of the weak dNd(i,i+1) NOE (Arg¹⁸to Pro¹⁹) suggested that aType I turn may be the preferred conformation (Dyson et al, J. Mol.Biol. 201:161–200 (1988)).

All three peptides also showed evidence of preferred conformers at thesequence Ser²⁶-Ile²⁷-Thr²⁸-Lys²⁹ (SEQ ID NO: 102). There were twoconsecutive daN(i,i+2) NOEs, between Ser²⁶ and Thr28 and between 1le²⁷and Lys²⁹, as well as medium range NOEs not shown in FIG. 2. The latterincluded a dbN(i,i+2) NOE between Ser²⁶ and Thr²⁸, and a dba(i,i+2) NOEbetween these same residues. The conformational preferences giving riseto these NOEs did not fit a typical secondary structure, and suggestedan unusual turn that placed the side-chain of Ser²⁶ in close proximityto the main-chain groups of Thr²⁸. This type of conformation has beendescribed as a kink in the context of a helical region (Osterhout et al,Biochemistry 28:7059–7064 (1989)).

A third conformational feature in the V3 segments of C4-V3RF,C4_(E9V)-V3RF(A) and C4_(E9G-V)3RF(A)occurred in the sequenceGly³⁰-Pro³¹-Gly³²-Arg³³ (SEQ ID NO: 66). In E9G the NOEs between theseresidues resembled the pattern described above that was consistent witha reverse turn (Dyson et al, J. Mol. Biol. 201:161–200 (1988)). Thisincluded a weak dNd(i,i+1) NOE between Gly³⁰ and Pro³¹, a weak ddN(i,i-I-i) NOE between Pro³¹ and Gly³², a weak dad(i,i+1) NOE between Gly³⁰and Pro³¹, a strong daN(i,i+1) NOE between Pro³¹ and Gly³², and adetectable daN(i,i+2) NOE between Pro³¹and Arg³³. In the C4-V3RFpeptide, the pattern of (i, i+1) NOE intensities was the same but nodaN(i,i+2) NOE was detected between Pro³¹ and Arg³³. Instead adaN(i,i+2) NOE was detected between Gly³⁰ and Gly³². And in C4-E9V V3RF,both daN(i,i+2) NOEs, Gly³⁰to Gly³² and Pro³¹ to Arg³³, were detected.These data raised the possibility that two independent turn-likeconforniational preferences occurred in this region of V3. The fact thata Pro³¹-Arg³³ daN(i,i+2) NOE was unambiguously absent in C4-V3RF, andthat a daN(i,I+2) NOE between Gly³⁰ and Gly³² was also unambiguouslyabsent in C4_(E9G)-V3RF(A), in spite of sequence identity in all threepeptides, may be related to the weak intensity of these NOEs. Beingclose to the level of noise intensity, there is a possibility that oneor both NOE signals on either side of the spectrum will not be detected,thus disallowing the given NOB to be scored as such.

Another region in V3 where conformational preferences could be inferredfrom NOEs occurs in residues Val³⁴-Ile³⁵-Tyr³⁶. In all three peptidesNOEs were observed between the upfield methyl resonance (˜0.67 ppm) ofVal³⁴ and the ring hydrogens, both dH and eH, of Tyr³⁶. Weaker NOEs arealso seen between the downfield methyl resonance (˜0.89 ppm) of Val³⁴and the ring hydrogens of Tyr³⁶. Further evidence of close proximitybetween the side-chains of Val³⁴ and Tyr³⁶ was the fact that the twomethyl resonances of the former had disparate chemical shifts, comparedto Val¹⁰, consistent with a ring-current shift induced by the aromaticside-chain of Tyr. One peptide, C4-V3RF(A) had another NOE in thisregion, daN(i,i+2) between Ile³⁵ and Ala³⁷, that was unambiguouslyabsent in the C4E9G-V3RF(A) and C4_(E9V)-V3RF(A) peptides. Thisobservation likely represented a poorly populated conformation, perhapsrelated to that which gives rise to the Val³⁴-Tyr³⁶ side-chaininteraction, or from an independent conformational propensity.

Substitution of Lys¹² with Glu yielded a poorly immunogenic peptide(C4_(K12E)-V3RF(A)) that, interestingly had solution propertiesdifferent from the other three peptides studied. Under the conditionsused for NMR studies of other C4-V3 peptides, the solution of theC4_(K12E)-V3RF(A) peptides was highly viscous, and viscosity increasedwith pH in the vicinity of pH 4, implicating ionization of the Glu¹²side-chain in this phenomenon. NMR spectra of K12E at 278 K in aqueousbuffer showed a much lower signal-to-noise ratio than the other threepeptides. Increasing the temperature to 318 K or decreasing the pH to3.5 yielded improved but still inadequate signal. Suitably high signalfor resonance assignment and NOE analysis was obtained at 318 K, pH 3.5,20% v/v trifluoroethanol (d₃). Even under this condition the NOEs forthe C4_(K12E)-V3RF(A) were less intense than for other peptides.

NOE connectivities in the C4 segment of C4_(K12E)-V3RF(A) (FIG. 2) showevidence of nascent helical turns in the region between Ile³ and Gly¹¹as inferred from dNN(i, i+2) and daN(i,i+2) NOEs. The stretch from Val¹⁰to Thr¹⁷ has two daN(i,i+3) and two dab(i,i+3) NOEs suggesting thepresence of a significant population with full helical turns. Within theV3 segment only two medium range NOEs are observed, both daN(i,i+2).Neither corresponds to NOEs observed in the other three peptides, butboth NOEs involve residues of the Ser²⁶-Ile²⁷-Thr²⁸ sequence, for whichthere is evidence of conformational preferences in the other threepeptides. A dbN(i,i+2) NOE between Ser²⁶ and Thr²⁸, observed inC4_(E9V)-V3RF(A)) and C4_(E9G)-V3RF(A), is also observed in the K12Epeptide. Also observed are NOEs between the side-chains of Val³⁴ andTyr³⁶. Hence the conformations giving rise to these two features are atleast partially preserved under the solution conditions employed forK12E. Differences in the V3 segment between K12E and all of the otherthree peptides include the absence of detectable daN(i, 1+2) NOE betweenPro¹⁹ and Asn²¹ and between Ser²⁶ and Thr²⁸. The failure to detect theseNOEs may be due to the overall weaker signals of this sample, or todepopulation of the relevant conformations by the solution conditions.

EXAMPLE 2

The peptides in Table 7 (SEQ ID NOS 31–60, respectively, in order ofappearance) have been studied in groups of 5 peptides as indicated inTable 9 (SEQ ID NOS 67–96, respectively, in order of appearance), andeach group of 5 peptides has been injected into each of three guineapigs in Freund's complete then incomplete adjuvant. After 4immunizations, the animals were bled, and heat inactivated serum waspooled from each animal or tested separately as indicated in Table 8,for the ability to neutralize HIV. Single numbers per group indicatethat the results are those of pooled sera from the group. Individualresults per animal indicate that each serum was tested individually.Table 8 shows that all the sera neutralized to varying degrees the Tcell line adapted HIV isolate MN and poorly neutralized the TCLA HIVisolate IIIB. Regarding the rest of the isolates in Table 8, all ofwhich are HIV primary isolates (89.6, BAL ADA, SF162, 5768, QH0515, PVO,JRFL, BX08, 6101, SS1196), Group C sera from C4-V3 subtype B peptidesneutralized 4/11(36%) and Group F sera from subtype B peptidesneutralized 5/11 primary isolates (45%). FIG. 4 shows that for the HIVCCR5 utilizing primary isolate, BAL, that the individual peptides in the5-valent mixture absorbed out the neutralizing activity against HIV BALto varying degrees, whereas the mixture of all the peptides completelyabsorbed out the neutralizing activity.

TABLE 8 Neutralization Of HIV-1 Isolates By Sera From Guinea PigsImmunized With C4-V3 Clade B Peptides Animal Immunogen HIVMN # HIVIIIB #SHIV89.6 # SHIV89.6 # HIVBAL* ADA* SF162* 5768* QH0515* PV0* JRFL* 477 A2,258 0  96 0 478 A 1,357 0 NA 35 0 0 90 0 0 0 0 479 A 4,632 68 NA 0 480B 1358 0 NA 0 481 B 7,774 0 NA 27 84 0 96 0 0 0 0 482 B 4,241 0  62 0483 C 969 0 112 95 484 C 806 0  20 97 84 0 99 0 0 0 0 485 C 542 0 226 80486 D 1,488 0 NA 0 487 D 2,184 0 NA 98 80 0 98 0 0 0 0 488 D 575 0 NA 0489 E 3,223 0 NA 88 490 E NA 0 NA 255 0 0 92 0 0 0 0 491 E 519 0 NA 81492 F NA 0 NA NA 493 F 910 0 NA 0 91 0 84 0 0 0 0 494 F 1,159 35 NA NAAnimal Immunogen BX08* 6101* SS1196* 477 A 478 A 0 0 85 479 A 480 B 481B 0 0 0 482 B 483 C 484 C 86 0 0 485 C 486 D 487 D 94 0 0 488 D 489 E490 E 0 0 0 491 E 492 F 493 F 91 94 88 494 F # Assay liters arereciprocal serum dilutions at which 50% of MT-2 cells were protectedfrom virus-induced killing as measured by neutral red uptake. *%reduction in p24 synthesis relative to the amount of p24 synthesized inthe presence of corresponding prebleed samples Values >80% are positive.NA = Not available

TABLE 9 G. Pig Immunization Protocol Part 2 Immunization with a group of5 peptides Peptide Name Peptide Sequence Code GP No. C4-V3 peptideC4-V3-23.38 KQIINMWQVVGKAMYA-RPIKIERKRIPLGLGKAFYTTK A 477, 478, 479C4-V3-11.85 KQIINMWQVVGKAMYA-RPNNNTRKSINIGPGRAFYTTG A C4-V3-34.29KQIINMWQVVGKAMYA-RPNNNTRKSIQIGPGRAFYTTG A C4-V3-1.481KQIINMWQVVGKAMYA-RPNNNTRKSIHIGPGRAFYTTG A C4-V3-3.323KQIINMWQVVGKAMYA-RPNNNTRKSINMGPGRAFYTTG A C4-V3-51.23KQIINMWQVVGKAMYA-RPSNNTRRSIHGLGRAFYTTG B 480, 481, 482 C4-V3-36.29KQIINMWQVVGKAMYA-RPNNNTRKGIHIGPGRTFFATG B C4-V3-57.20KQIINMWQVVGKAMYA-RPNRHTGKSIRMGLGRAWHTTR B C4-V3-35.29KQIINMWQVVGKAMYA-RPNNNTRKRISLGPGRVYYTTG B C4-V3-46.26KQIINMWQVVGKAMYA-RPDNTIKQRIIHIGPGRPFYTT B C4-V3-69.18KQIINMWQVVGKAMYA-RPNNNTRKSIRIGPGRAVYATD C 483, 484, 485 C4-V3-72.18KQIINMWQVVGKAMYA-RPNNNTRKGINIGPGRAFYATG C C4-V3-70.18KQIINMWQVVGKAMYA-RPNNNTRKRIRIGHIGPGRAFYATG C C4-V3-62.19KQIINMWQVVGKAMYA-RPNNNTRKSIHIGPGRAFYATE C C4-V3-74.17KQIINMWQVVGKAMYA-RPNNNTRKRMTLGPGKVFYTTG C C4-V3-82.15KQIINMWQVVGKAMYA-RPNNNTRRSIPIGPGRAFYTTG D 486, 487, 487 C4-V3-113.1KQIINMWQVVGKAMYA-RPNNNTRRSIHLGMGRALYATG D C4-V3-89.14KQIINMWQVVGKAMYA-RPSINKRRHIHIGPGRAFYAT D C4-V3-85.15KQIINMWQVVGKAMYA-RPNNNTRKSIHIAPGRAFYTTG D C4-V3-122.9KQIINMWQVVGKAMYA-RPNYNETKRIRIHRGYGRSFVTVR D C4-V3-170.6KQIINMWQVVGKAMYA-RPNNNTRRSVRIGPGGAMFRTG E 489, 490, 491 C4-V3-146.8KQIINMWQVVGKAMYA-RPGNNTRRRISIGPGRAFVATK E C4-V3-163.7KQIINMWQVVGKAMYA-RLYNYRRKGIHIGPGRAIYATG E C4-V3-125.9KQIINMWQVVGKAMYA-RPNNNTRRRISMGPGRVLYTTG E C4-V3-162.7KQIINMWQVVGKAMYA-RPGNNTRGSIHLHPGRKFYYSR E C4-V3-396.2KQIINMWQVVGKAMYA-RPNNNTRRNIHIGLGRRFYAT F 492, 493, 494 C4-V3-144.8KQIINMWQVVGKAMYA-RPDNNTVRKIPIGPGSSFYTT F C4-V3-365.2KQIINMWQVVGKAMYA-RPSNNTRKGIHLGPGRAIYATE F C4-V3-513.2KQIINMWQVVGKAMYA-RPSNNTRKGIHMGPGKAIYTTD F C4-V3-1448.1KQIINMWQVVGKAMYA-RPGNTTRRGIPIGPGRAFFTTG F

It is important to be able to use T helper detenninants with the V3portion of the peptides shown in Table 7, both to expand the T helperactivity in the immunogen, and in case any of the T helper peptidesshould be found to have any deleterious effects in the course of humantrials. For example, it has recently been found in vitro that in cultureof HIV and T cells, that the C4 portion of the C4-V3 peptide can augmentHIV induced syncytium formation. However, peptides of this generaldesign have been studied in vitro in HIV-infected humans (AIDS 12:1291–1300, 1998) and no subjects developed a ≧10 fold change in plasmaHIV RNA levels from baseline. Moreover, the primary use of thesepeptides is as an immunogen in HIV—subjects as a preventive vaccine, andnot in doses that one would consider for therapy, which would be inmilligram amounts daily. A T helper determinant from HIV gag, termedGTH1 with the sequence of Y K R W I I L G L N K I V R M Y S (SEQ ID NO:97) has been conjugated to the V3 of HIV MN and found to induce anti-HIVMN titers of 1:3200. Similarly, GTH1 conjugated to a V3 sequence of aHIV primary isolate DU179 induced antibodies that neutralized IrHV MN(1:192) and neutralized the HIV primary isolate JR-FL (90% p24 reductionin PBMC cultures). Thus, the GTHI T helper sequence can substitute forthe C4 sequence in the peptides in Table 7.

Finally, a panel of monovalent serum from individual guinea pigsimmunized with each of the peptides in Table 7 has been screened.Whereas most of the peptides in the list only induced neutralizingantibodies that neutralized 0 to 6 out of 19 primary isolates, 5peptides were found that neutralized from 14 to 19 out of 19 primaryisolates tested. These peptides were C4-V3 36.29, C4-V3 34.29, C4-V362.19, C4-V3 74.17, and C4-V3 162.7. The sequences of these peptides areall listed in Table 7.

Thus, sufficient breadth has been observed both in mixtures of C4-V3peptides and in select individual peptides for the immunogen to bepractical with regard to induction of neutralizing antibodies againstHIV primary isolates. By performing the same immunization studies withthe similarly designed HIV subtype (clade) C peptides in Table 6, that asimilar immunogen(s) can be developed for HIV subtype C viruses.

While individual peptides can be used to achieve the breadth ofneutralizing activity needed to protect against HIV primary isolates,advantageously, mixtures of multiple peptides are used, such as thecombination of group C, or group F or the combination of C4-V3 36.29,C4-V3 34.29, C4-V3 62.19, C4-V3 74.17, and C4-V3 162.7 peptidesdescribed above.

EXAMPLE 3 HIV-1 Clade B V3-Based Polyvalent Immunogen

Anti-HIV gp120 V3 antibodies can neutralize some HIV primary isolates((Hioe et al, Internat. Immunology 9:1281 (1997), Liao et al, J. Virol.74:254 (2000), Karachmarov et al, AIDS Res. Human Retrovirol. 17:1737(2001), Letvin et al, J. Virol. 75:4165 (2001)). The hypothesis forthese studies was that sequence variation found among HIV primaryisolates need not reflect the diversity of HIV serotypes, and antibodiescan cross-react with groups of similar viruses. Data from comparison ofNMR structures of several V3 loops and their immunogenicity patternsindicate that there are conserved higher order structures of the V3 thatare similar in antigenicity regardless of primary amino acidheterogeneity (Vu et al, J. Virol. 73:746 (1999)).

1514 unique clade B V3 sequences in the Los Alamos National LaboratoryHIV Database were analyzed by the following methods. Short antigenicdomains were organized by protein similarity scores usingmaximum-linkage clustering (Korber et al, J. Virol. 68:6730 (1994)).This enabled visualization of clustering patterns as a dendritogram, andthe splitting pattern in the dendritogram could be used to defineclusters of related sequences. This method allows the use of severaldifferent amino acid scoring schemes. The amino acid substitution matrixof Henikoff and Henikoff was used which was designed to give amino acidsubstitutions well tolerated in conserved protein structural elements ahigh score, and those that were not, a low score (Henikoff and Henikoff,Protein Structure Function and Genetics 17:49 (1993)). Based on theseclustering patterns, a variant was selected that was most representativeof each group. Excluded were very rare, highly divergent sequences, andfavored were sequences found in many different individuals. This methodallowed for most of the unique V3 sequences to be within one or twoamino acids from at least one of the peptides in the cocktail. Thus,1514 lade B V3 sequences were clustered into 30 groups. The consensuspeptide of each group was synthesized, purified to homogeneity by HPLCand confirmed to be correct by mass spectrometry. Each peptide wasimmunized into a guinea pig (GP) in Incomplete Freunds Adjuvant (IFA),and each sera was tested after the fifth immunization by a singleinfection cycle neutralization assay preformed by ViroLogics, South SanFrancisco, Calif., or by a fusion from without HIV fusion inhibitionassay using aldrithiol-2 inactivated HIV_(ADA), HIV_(MN) and HIV_(AD8)virons (Rosio et al, J. Virol. 72:7992 (1998)).

The criteria established for acceptable neutralization of primaryisolates was the ability of a serum to neutralize at least 25% of theHIV primary isolates tested. Using these criteria, 7 peptides were foundthat induced neutralizing antibodies against >25% of isolates tested.One of these peptides, peptide 62.19, neutralized 19/19 HIV primaryisolates tested, even when the criteria were increased to greater than80% neutralization vs. 50% neutralization (see FIG. 5 and Table 11).

When the sequences of 6 peptides that induced no (0/19) neutralizationof the 19 primary HIV isolates were evaluated, it was found that theywere all unusual sequences at the tip of the V3 loop, with sequencessuch as GLGR (SEQ ID NO: 98), GPGG (SEQ ID NO: 99), GLGK (SEQ ID NO:100V GLGL (SEQ ID NO: 101), and GLGR (SEQ ID NO: 98) present (see Table10: (SEQ ID NOS 103–108, respectively, in order of appearance)). Only 1of the 19 isolates tested had one of the these V3 sequences, a GPGG (SEQID NO: 99) sequence, that was not neutralized by the serum from theGPGG-immunized (SEQ ID NO: 99) guinea pig. Therefore, one serologicdefined group of Clade B HIV isolates may be defined by the primaryamino acid sequences at the tip of the loop of GLGR (SEQ ID NO: 98),GPGG (SEQ ID NO: 99), GLGK (SEQ ID NO: 100), GLGL (SEQ ID NO: 101).

TABLE 10 Sequences of Peptides That Induced No Neutralization at 50%Inhibition (All Dilutions) Criteria GP No. Peptide No. V3 Sequence(s)447 C4-V3 396.2 RPNNNTRRNIHIGLGRRFYAT 448 C4-V3 170.6RPNNNTRRSVRIGPGGAMFRTG 451 C4-V3 23.38 RPIKIERKRIPLGLGKAFYTTK 458 C4-V351.23 RPSVNNTRRSIHMGLGRAFYTTG 404 C4-V3 57.20 RPNRHTGKSIRMGLGLAWHTTR 432396.2/170.6 RRNIGIGLGRRF     RRSVRIGPGGAM

TABLE 11 Sequences of Peptides That Best Neutralized Clade B Isolates at50% Inhibition (All Dilutions) Criteria GP No. Peptide No. V3Sequence(s) 436 69.18/146.8 RKSIRIGPGRAV    RRRISIGPGRAF 442 1.481/85.15RKSIHIGPGRAF    RKSIHIAPGRAF 460(B) C4-V3 36.29 RPNNNTRKGIHIGPGRTFFATG465(A) C4-V3 11.85 RPNNNTRKSINIGPGRAFYTTG 466(A) C4-V3 34.29RPNNNTRKSIQIGPGRAFYTTG 467(A) C4-V3 1.481 RPNNNTRKSIHIGPGRAFYTTG 469(C)C4-V3 62.19 RPNNNTRKSIHIGPGRAFYATE 472(C) C4-V3 74.17RPNNNTRKRMTLGPGKVFYTTG 475(E) C4-V3 162.7 RPGNNTRGSIHLHPGRKFYYSR

When the peptide sequences that induced neutralization of >25% ofprimary isolates were examined, it was found that the sequences were allsimilar and were all clustered around the Glade B V3 consensus sequenceof IHIGPGRAFYTTG (SEQ ID NO: 118) (see Table 11: SEQ ID NOS 109–117,respectively, in order of appearance). However, not all peptides withthis type of sequence induced good neutralizing antibodies—15 peptideshad this type of sequence and did not induce good neutralizingantibodies. Thus, a “computer guided proteomic screen of the V3 loop”has been performed and V3 peptides have been identified that expresshigher order conformers that mirror the native functionally active motifof the V3 that is both available and capable of being bound byneutralizing antibodies. In particular, peptide 62.19 inducedneutralizing antibodies against 19 of 19 HIV isolates.

Expression of the consensus B V3 sequences in Table 11, and expressionof certain of the unusual V3 sequences in Table 10, can define a“bivalent” clade B immunogen for use world wide where those sequencesare present in the resident HIV quasispecies. Immunization with areplicating vector, expressing partial or entire (C to C) segments ofthese V3 loops, can be used to induce long lasting immunity to HIV.

All documents cited above are hereby incorporated in their entirety byreference.

1. An isolated polypeptide comprising the sequence of SEQ ID NO:115. 2.A composition comprising a polypeptide comprising the sequence of SEQ IDNO:115 and a carrier.
 3. The isolated polypeptide of claim 1, whereinsaid polypeptide further comprises a T-helper epitope.
 4. The isolatedpolypeptide of claim 3, wherein said T-helper epitope is an HIV T-helperepitope.
 5. The isolated polypeptide of claim 4, wherein said T helperepitope comprises residues of the C4 domain of gp120. 5
 6. A method ofinducing the production of antibodies in a mammal comprisingadministering to said mammal an amount of said polypeptide according toclaim 1 sufficient to effect said induction.
 7. The method of claim 6wherein said polypeptide further comprises a T-helper epitope.
 8. Themethod of claim 7, wherein said T-helper epitope is an HIV T-helperepitope.
 9. The method of claim 8 wherein said HIV T-helper epitopecomprises residues of the C4 domain of gp120.